Botulinum neurotoxins (BoNT) are a high priority of the national biodefense initiative because of their ability to target the cytosol of motor neurons at extremely low doses, even when delivered via mucosa. Extensive work characterizing the molecular mechanism of BoNT action has created the potential to use atoxic derivatives of BoNT as neuron-targeting delivery vehicles for diverse therapeutic agents, including agents directed against neurotropic toxins and organisms, and antidotes to BoNT itself. We are therefore designing and testing a genetic engineering platform for producing atoxic BoNT derivatives that target the neuronal cytosol after oral or inhalational administration. Our genetic constructs facilitate manipulation and modification of BoNT gene segments, and enable expression of recombinant, full-length, atoxic BoNT derivatives that retain the native toxin's key structural features, disulfide bonding and targeting properties. We here propose expressing and purifying a prototype atoxic BoNT derivative in quantities, sufficient for biological testing, to ensure that any residual toxicity has been eliminated, and that the capacity to deliver atoxic light chains to the neuronal cytosol in reasonable quantities is retained. Subsequently, we will use this genetic engineering platform to produce a series of BoNT derivatives in which a fluorescent marker has been built directly into the derivative's amino acid sequence. These enable BoNT trafficking pathways to be evaluated using direct fluorescence microscopy, and will compliment data from indirect immunodetection techniques. Four fluorescent BoNT derivatives are proposed, differing in the size and structure of the flurophor, and the size of the LC polypeptide segment retained. Experiments comparing the intra-cellular trafficking of the fluorescent BoNT derivatives to each other, and to the prototype non-fluorescent BoNT derivative, will provide important information about the role of the LC catalytic domain in BoNT targeting, and establish limits to the size and structure of cargo molecules that can transported to the neuronal cytosol using a BoNT-based mechanism. One of the fluorescent BoNT derivatives is of particular interest because the fluorophor is enzymatically coupled to a short amino acid sequence inserted at the N-terminus of the LC construct, but which should be otherwise identical to native BoNT ad. It therefore provides a new tool to study the molecular mechanism of BoNT trafficking that resolves ongoing problems associated with the non- selective attachment of flurophors to BoNT using chemical methods. [unreadable] [unreadable] Project Narrative Botulinum neurotoxins (BoNT) are a high biodefense priority because they target motor neurons at extremely low doses, even when delivered via mucosa. We are therefore developing a genetic engineering platform that can take advantage of BoNT's neuron-targeting properties to deliver diverse therapeutic agents to the neuronal cytosol, including antidotes to BoNT itself. This proposal will evaluate the molecular features required for neuron targeting, and produce a new class of fluorescent BoNT derivatives that will be broadly useful to study the molecular mechanisms of BoNT trafficking. [unreadable] [unreadable] [unreadable]